Project Summary The last 10 years have witnessed explosive advances in our understanding of how the organization of chromatin, the association of DNA with histones and the vast number of non-histone regulatory proteins, controls the expression of specific genes in brain. Likely prominent among such regulatory mechanisms are covalent modifications of histones, along with the writers, erasers, and readers of these modifications. Much of the work delineating these mechanisms has contributed to the notion that a `histone code' is a central determinant of a gene's activity and its potential to be activated or repressed in response to a subsequent stimulus (e.g., elicit drug exposure). Indeed, increasing evidence has suggested the significance of histone modifications in behavioral models, although we are still at the earliest stages of examining all of the many chromatin changes potentially involved. Based, in part, on our work in the field of addiction neuroepigenetics, we now have substantial evidence for profound regulation of chromatin mechanisms in rodent models and in human postmortem brain: chronic psychostimulant exposure alters covalent modifications of histones that may powerfully influence addiction vulnerability; and chronic cocaine alters the functionality of specific ?writer? and ?eraser? enzymes that control these modifications, as well as the ?reader? proteins that translate the modifications into functional responses. Although exciting, nearly all data to date indicating roles for histone modifications in addiction plasticity have been deduced from studies involving manipulations of associated enzymes and/or correlational sequencing analyses, with little to no attention paid to the development of novel methodologies that would allow for direct, causal manipulations of histone modifications in brain. Therefore, this Cutting Edge Basic Research Award (CEBRA) proposal aims to fully apply a novel intein- based chemical methodology for directly introducing specific histone modifications (or combinations thereof) in neurons to directly assess their impact on transcriptional plasticity and addiction related behaviors. To do so, we will focus on two forms of histone modifications (permissive?H3.3 pan-acetyl and repressive?H3.3K9me2) that have previously been implicated in cocaine responsiveness, both by our group and others. Since our initial submission last year, we have now chemically optimized the efficiency of the intein system for use in brain and provide robust preliminary data toward this effect. Thus, we now aim to: 1) assess the impact of promoting such modifications (which, based upon previous findings, are expected to result in opposing behavioral outcomes) on relapse related behaviors in male vs. female rats; and 2) fully examine the transcriptional impact of directly introducing specific histone modifications in chromatin in neurons using next generation sequencing, both in the context of cocaine self-administration (SA) and in drug naive animals (e.g., food SA). We feel that this novel methodology will have a broad impact on future studies related to neuroepigenetic plasticity in addiction, and will be highly applicable to numerous other research areas. !